General description - Mitosis and meiosis in the germline - Germline programmed cell deaths - Oocyte maturation, ovulation and fertilization - Germline development - Spermatogenesis and spermiogenesis - Back to Contents
The hermaphrodite germ line produces both male and female gametes, sperm and oocytes, respectively. Oocytes are produced throughout adult life; sperm (spermatozoa) are generated during L4 then used in adulthood to fertilize oocytes. The adult germ line exhibits distal-proximal polarity with a mitotic cell population located at the most distal end of the gonad (DG) and meiotic cells extending proximally. Among the meiotic cells, there is also a gradient of meiotic progression with progressive stages of prophase of meiosis I extending from the distal arm, around the loop into the proximal arm of the gonad. Gametogenesis occurs in the proximal part of the gonad arm (GerFIG1) .
The distal germ line is a syncytium. Germ cells have incomplete borders and are connected to one another via a central canal, the rachis (GerFIG1, 2A, 2B ; Hirsh et al., 1976). Part of the distal gonad is not covered by the somatic tissues (the "bare region") and instead is ensheathed only by the gbl (gonadal basal lamina) that covers the rest of the gonad (Hall et al., 1999; GerFIG2A, B). At the base of each germ cell, and covering the rachis, is a thicker extracellular matrix. This thicker matrix contains hemicentin and is thought to reinforce and stabilize the opening of the germ cells to the rachis (GerFIG2C, 2D). The endpoint of the rachis may differ as the animal ages. According to McCarter et al. (1997), the rachis terminates within the proximal gonad, just past the loop in young adults and in older adults terminates in the distal gonad, although still near the loop. Oocytes may retain a vestigial connection to the rachis even after moving well past its apparent endpoint (J. White, pers. comm.) so that a maturing oocyte in the proximal arm might retain a thin, cryptic arm reaching through the loop to the distal rachis.
The most distal end of the adult gonad contains a stem cell population and is referred to as the mitotic zone. As germ cells move away from the influence of the distal tip cell (DTC; see Part II - somatic gonad), they enter meiosis I, proceeding through prophase I to diakinesis (GerFIG3A-E; Hirsh et al., 1976; reviewed in Hubbard and Greenstein, 2000; Hansen et al., 2004).
The mitotic zone of the adult is approximately 20 cell diameters in length, extending the from the DTC to the transition zone (described below; Crittenden et al., 1994; GerFIG3B). With DAPI staining at the light microscopic level, M-phase nuclei can be distinguished from the rest of the cell cycle. In these preparations, most nuclei are relatively uniform and, at any given time, have a hazy fluorescence in the center and brighter circumferential staining. Condensed chromatin appears as nuclei enter pro-metaphase. The average number of M-phase nuclei visible in any given mitotic zone in the adult is low about 2 nuclei per arm (Maciejowski and Hubbard, unpublished). At the EM level, mitotic germ cells are uniform in size and appearance (GerFIG2C). Each cell is roughly cuboidal with a large nucleus. The cytoplasm contains a few mitochondria, limited RER and some free ribosomes. The rachis itself is also filled with RER and ribosomes but contains more mitochondria. The transition zone is characterized by germ cells entering the early phases of meiotic prophase (leptotene and zygotene) and is defined as the area between the distal-most transition nucleus and the proximal-most transition nucleus (Hansen et al., 2004). A change in the nuclear morphology can be visualized with DAPI: nuclei are condensed and crescent-shaped (GerFIG3C).
After moving through the transition zone, germ cells progress into pachytene and gradually grow. Pachytene nuclei are characterized by a distinctive bowl of spaghetti morphology (GerFIG3D). Exit from pachytene requires activation of a MAPKinase pathway, thought to be triggered by a signal from overlying gonadal sheath (Church et al., 1995; McCarter et al., 1997). Progression of nuclei into diplotene occurs in the loop and cells become organized in single file as they enter the proximal arm (GerFIG3E).
In the proximal arm oocytes progress to diakinesis (GerFIG3E,3F) where they arrest until oocyte maturation, a prerequisite to ovulation and fertilization (see below). Oocytes enlarge to fill the entire space within the gonad arm, greatly increasing their cytoplasmic contents and the size of their nucleus. The number of mitochondria, RER, free ribosomes and types of endosomal organelles also increases. Oocytes closest to the spermatheca begin to swell with yolk granules, formed by endocytoses of yolk protein that has traversed the sheath pores from the pseudocoelom (Grant and Hirsh, 1999; Hall et al., 1999; see Part II - somatic gonad). Oocytes closest to the spermatheca are the most advanced in maturity.
In addition to oocyte and sperm fate, PCD (Programmed Cell Death) represents a major cell fate among adult germ cells. It is estimated that approximately one half of all potential oocytes are eliminated in the adult hermaphrodite during progression through prophase of meiosis I. Most cell deaths occur near the loop region of the gonad arm, the region containing pachytene stage germ cells (see GerFIG3A,3D). It has been proposed that these excess germ cells may serve as a nurse cell population, providing proteins and other cytoplasmic components to surviving germ cells (Hengartner, 1997). EM and light microscopy analyses of dying cells reveal that cell deaths occur by apoptosis (GerFIG4; Gumienny et al., 1999). As in somatic tissues, cell death execution depends on ced-3, ced-4 and ced-9 function. However, there is also genetic evidence suggesting that somatic and germ cell death mechanisms may not be entirely identical (Hengartner et al., 1992; Gumienny et al., 1999). Cell corpses are engulfed by the overlying gonadal sheath cells (Gumienny et al., 1999).
Oocyte maturation takes place in the oocyte closest to the spermatheca, just prior to ovulation, and is stimulated by sperm-derived MSP (Major Sperm Protein) (Miller et al, 2001). During maturation, NEBD (Nuclear Envelope Break Down; also known as GVBD for Germinal Vesicle Break Down) occurs, the nucleus becomes less obvious and cortical rearrangements cause the oocyte to become more spherical. Chromosome arrangement changes as bivalents leave diakinesis and begin to align onto the metaphase plate (GerFIG5A,5B; Ward and Carrel, 1979; McCarter et al.,1999).
Ovulation follows oocyte maturation (see ovulation movie by Miller et al, 2001). Signals from the maturing oocyte and MSP stimulate the rate and intensity of sheath contraction from a basal rate of 10-13 contractions/min to approximately 19 contractions/min (McCarter et al.,1999; Miller et al, 2001; 2003). Oocyte maturation also stimulates distal spermathecal dilation through lin-3/let-23 RTK pathway activation and IP3 signaling (see Part II - somatic gonad; Clandinin et al., 1998; McCarter et al.,1999; Bui and Sternberg, 2002). The dilated spermatheca is pulled over the oocyte by the contracting sheath. The spermatheca then closes. The oocyte is immediately penetrated by a sperm and fertilized. Cell-cell recognition between gametes during this process is mediated by SPE-9, a sperm-specific, EGF-repeat-containing transmembrane protein (Singson et al., 1998). Cytoplasmic streaming in the oocyte accompanies fertilization, meiosis is completed, and egg shell secretion commences (Ward and Carrel, 1979; Singson, 2001). The newly formed embryo then passes from the spermatheca to the uterus via the spermatheca-uterine valve (sp-ut).
Germline development spans L1 to early adulthood. All germ cells are descended from either Z2 or Z3 (Schedl, 1997; Hubbard and Greenstein, 2000). Key events in germline development are summarized in GerFIG6 (for animation see Hubbard lab web page). In contrast to somatic lineage development in C. elegans, germline cell divisions appear to be variable with respect to their timing and planes of division (Kimble and Hirsh, 1979) and hence the precise lineal relationships between these cells is not known. In L4, approximately 37 meiotic cells per arm at the most proximal end of the germ line commit to sperm development. Subsequently, the germ line switches from making sperm to making oocytes for the remainder of development and throughout adulthood. This switch between male and female cell fate results from germ line modulation of the sex determination pathway activity (Kuwabara and Perry, 2001). Germline development depends on interactions with the overlying somatic gonad. Somatic gonad cells, or their precursors, affect the timing and position of the germline mitosis/meiosis decision, exit from pachytene, gametogenesis and male germline fate during germline sex determination (Kimble and White, 1981; Seydoux et al., 1990; McCarter et al., 1997; Rose et al., 1997; Pepper et al, 2003; Killian and Hubbard, 2004).
Animation of Hermaphrodite Gonadogenesis. Press "Play" to start,"Pause" to stop or click boxes to view specific stages. Note, movie does not follow the color code used in GerFIG6 above. Yellow, germ nuclei; Red , DTC nuclei; Purple, other early somatic cell nuclei. As germline development proceeds, mitotic nuclei remain yellow, while green represents meiotic stages (light green for early stages (leptotene and zygotene) and darker for later stages (pachytene)). Blue, spermatocytes; Dark Blue, mature sperm; Pink, oocytes. (note: one gonad arm is depicted but the other develops in the same way). Kindly provided by E. J. Hubbard. Animation by Rob Stupay © 2003.
The germ line of each gonad arm produces about 150 sperm during L4 (GerFIG6,7A; L'Hernault, 1997). Approximately 37 diploid germ cells/arm form 1° spermatocytes while still attached to the rachis. After pachytene, spermatocytes detach from the rachis and complete meiosis generating haploid spermatids. This process of spermatid formation is called spermatogenesis (GerFIG7B-D; Ward et al., 1981).
Developing spermatocytes contain a large number of specialized vesicles called FB-MOs (Fibrous Body-Membranous Organelle) (GerFIG8A,8B). These organelles contain proteins required in the future spermatid and spematozoon, including MSP (Ward and Klass,1982). During development, the FB-MOs partition with the portion of the spermatocyte destined to become the future spermatid (Ward, 1986). The residual body (GerFIG7D) acts as a depot for proteins and organelles no longer required by the developing spermatid (L'Hernault, 1997; Arduengo et al., 1998; Kelleher et al., 2000).
Spermatogenesis takes place within the proximal gonad (GerFIG7B; 9A). The spermatids formed are pushed into the spermatheca by the first oocyte during the first ovulation. In the spermatheca an unknown signal induces these sessile spermatids to undergo morphogenesis into mature, amoeboid spermatozoon (sperm) (GerFIG9B; Nelson and Ward, 1980; Ward et al., 1983). This process of activation is known as spermiogenesis (for movies of spermatogenesis and spermiogenesis see Ward lab website).
Maturing spermatids and spermatozoa have highly condensed nuclei (N) and tightly packed mitochondria (M) (GerFIG9A, 9B). In spermatids, MOs (now lacking FB) locate near the cell periphery (GerFIG8C,9A). During spermatid activation, MOs fuse with the plasma membrane releasing their contents (primarily glycoproteins) onto the cell surface. A fusion pore is generated on the cell surface by the MO collar (GerFIG9B). Mutants affected in MO fusion produce sperm with defective motility suggesting that MO content enhances sperm mobility (Ward et al., 1981; Roberts et al, 1986; Achanzar and Ward, 1997). Spermatid activation also involves the formation of a foot or pseudopodium. In contrast to sperm in other phyla, C. elegans sperm lack flagella. Pseudopodia allow spermatozoa to attach to the walls of the spermathecal lumen and to crawl. This motility is driven by dynamic polymerization of MSP. In addition to an intracellular cytoskeletal function, MSP contains sequences that mediate extracellular signaling (described above; Miller et al, 2001; Italiano et al., 1996; Roberts and Stewart, 2000; for movies of sperm crawling and motility see Ward lab website).
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